A thrombin-triggered self-regulating anticoagulant strategy combined with anti-inflammatory capacity for blood-contacting implants

Figures

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  Fig. 1.. The functional model and preparation process of the thrombin-triggered anticoagulant strategy combined with anti-inflammatory capacity for blood-contacting implants. Ox-HA, oxidized hyaluronic acid; RIVA, rivaroxaban; EGCG, epigallocatechin gallate; PEI-Tpl, Tempol-grafted polyethylenimine; R@NG, nanogels loaded with RIVA.

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  Fig. 2.. Characterization of nanogels and coating. (A) Accumulated RIVA release from thrombin-sensitive nanogels in PBS with different thrombin concentrations. (B) SEM and (C) AFM images of R@NG/Tpl and R@NG/EGCG/Tpl. (D) UV-vis spectra of R@NG/Tpl and R@NG/EGCG/Tpl on a quartz substrate. (E) Surface zeta potential of R@NG/Tpl and R@NG/EGCG/Tpl at pH 7.4. Real-time monitoring of (F) R@NG/Tpl and (G) R@NG/EGCG/Tpl assembly with QCM-D. (H) ΔD-ΔF curve of nanogel layers in R@NG/EGCG/Tpl coating.

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  Fig. 3.. Stability and anti-inflammatory capacity of the coating. (A) Pulsatile release profile of RIVA from R@NG/EGCG/Tpl under phasic exposure to the thrombin concentration of 10 U/ml. (B) RIVA loss rate of R@NG/EGCG/Tpl immersed in PBS at 37°C over 28 days. SEM images of R@NG/EGCG/Tpl after being immersed in PBS at 37°C for 7 days (C) and 14 days (D). (E) DPPH radical scavenging capability of the coatings. Fluorescence images of the (F) morphology and ROS generation of 160 nM PMA-treated THP-1 cells on different samples after 48 hours. Scale bar, 50 μm. (G) TNF-α and (H) IL-10 levels expressed by THP-1 cells detected by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (t test, error bars are defined as SD).

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  Fig. 4.. Anticoagulation and cytotoxicity. (A) Platelet adhesion and activation on the surface of samples. (B) Number of platelets adhering to the surface of samples. (C) Fluorescence images of HUVECs stained by calcein and (D) cell viability of HUVECs cultured on the samples after 24 and 72 hours. Scale bar, 200 μm. (E) Fluorescence images of HUASMCs stained by calcein. Scale bar, 500 μm. (F) Cell viability and (G) mean radius ratio of HUASMCs on the samples after 24 and 72 hours. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (t test, error bars are defined as SD).

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  Fig. 5.. Performance of the coating on biological valve leaflets. (A) Representative stress-strain curves, (B) thermal shrinkage temperature, and (C) weight loss ratio of biological valve leaflets. (D) Morphology of biological valves with and without R@NG/EGCG/Tpl modification observed by SEM. Scale bar, 1 μm. (E) Schematic diagram of the ex vivo arteriovenous shunt model. (F) SEM images and (G) thrombus weight of biological valve leaflets after 1-hour ex vivo arteriovenous shunt assay. Scale bar, 10 μm. (H) Photographs and (I) quantitative analysis of H&E staining of biological valve leaflets subcutaneously implanted in Sprague-Dawley rats. Scale bar, 200 μm. (J) Photographs of immunofluorescence analysis of biological valve leaflets. Scale bar, 50 μm. Capsules were traced by lines, and biological valves were marked by stars. (K) Quantitative data of immunofluorescence analysis of the capsule surrounding biological valve leaflets. Calcium deposits on biological valve leaflets characterized by (L) alizarin red staining and (M) ICP-OES. Scale bar, 200 μm. *P < 0.05, **P < 0.01, and ****P < 0.0001 (t test, error bars are defined as SD).

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  Fig. 6.. Characterization, hemocompatibility, anticoagulation, and endothelialization of vascular stents modified with the coating. (A) SEM images of PLA vascular stents before and after R@NG/EGCG/Tpl modification. Scale bar, 10 μm. (B) SEM images of R@NG/EGCG/Tpl-modified vascular stent after dilation. Scale bars, 300 and 10 μm. (C) SEM images and (D) thrombus weight of vascular stents after blood circulation in ex vivo arteriovenous shunt assay for 1 hour. Scale bar, 10 μm. (E) Photographs and (F) quantitative analysis of histomorphological evaluation of subcutaneously implanted vascular stents. Scale bar, 200 μm. The fibrous encapsulation was marked by the red lines. (G) Photographs and (H) quantitative data of immunofluorescence analysis of subcutaneously implanted vascular stents. Scale bar, 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test, error bars are defined as SD).

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  Fig. 7.. In vivo histocompatibility of the vascular stent modified with the coating. (A) Morphology of the luminal vascular stents after implantation in the abdominal aorta of New Zealand rabbits observed by SEM. Scale bars, 200 and 25 μm. The representative cells oriented in the direction of the blood flow were marked with orange arrows. (B) Immunofluorescence analysis of the vascular stents after implantation in the abdominal aorta of New Zealand rabbits. Scale bar, 100 μm. Quantitative analysis of (C) cell area ratio, (D) CD31 expression, and (E) endothelial nitric oxide synthase (eNOS) expression of the vascular stents. (F) Histological and immunohistochemical analysis of the vascular stents after implantation in the abdominal aorta of New Zealand rabbits. Scale bars, 500 and 100 μm. Quantitative analysis of (G) neointimal stenosis, (H) CD68 expression, and (I) α-SMA expression of the abdominal aorta implanted with vascular stents. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (t test, error bars are defined as SD).